Natural and Derivative Brevetoxins Historical Background by xny14254


									                                                                                        Brevetoxins | Mini-Monograph
Natural and Derivative Brevetoxins: Historical Background, Multiplicity,
and Effects
Daniel G. Baden, Andrea J. Bourdelais, Henry Jacocks, Sophie Michelliza, and Jerome Naar
University of North Carolina at Wilmington, Center for Marine Science, Wilmington, North Carolina, USA

                                                                                                           structures being determined by comparison of
 Symptoms consistent with inhalation toxicity have long been associated with Florida red tides,            spectra with those of the two parent toxins,
 and various causal agents have been proposed. Research since 1981 has centered on a group of              PbTx-1 and PbTx-2.
 naturally occurring trans-fused cyclic polyether compounds called brevetoxins that are produced
 by a marine dinoflagellate known as Karenia brevis. Numerous individual brevetoxins have been              Toxin Structure–Activity
 identified from cultures as well as from natural bloom events. A spectrum of brevetoxin derivatives        Relationships
 produced by chemical modification of the natural toxins has been prepared to examine the effects           By molecular modeling, Rein et al. (1994a,
 of functional group modification on physiologic activity. Certain structural features of natural and       1994b) and Gawley et al. (1995) determined
 synthetic derivatives of brevetoxin appear to ascribe specific physiologic consequences to each           that the structures of both PbTx-1 and PbTx-2
 toxin. Differential physiologic effects have been documented with many of the natural toxins and          are relatively linear with a bend approximately
 derivatives, reinforcing the hypothesis that metabolism or modification of toxin structures modu-          mid-molecule, possess a lactone functionality in
 lates both the specific toxicity (lethality on a per milligram basis) and potentially the molecular       the A-ring, have a strictly rigid region in the
 mechanism(s) of action. A series of naturally occurring fused-ring polyether compounds with               terminal four rings, possess a side chain allow-
 fewer rings than brevetoxin, known as brevenals, exhibit antagonistic properties and counteract           ing modest modification at the molecules’ ter-
 the effects of the brevetoxins in neuronal and pulmonary model systems. Taken together, the               mini, and have a spacer region that separates
 inhalation toxicity of Florida red tides would appear to depend on the amount of each toxin pre-          the rigid region from the A-ring lactone. All
 sent, as well as on the spectrum of molecular activities elicited by each toxin. Toxicity in a bloom      natural brevetoxins and synthetic derivatives
 is diminished by the amount brevenal present. Key words: brevenal, brevetoxin, Florida red tide,          with full activity possess all these features.
 immunotoxicant, Karenia brevis, neurotoxin, PbTx, polyether, pulmonary toxicant. Environ                  Numerous publications describe many of the
 Health Perspect 113:621–625 (2005). doi:10.1289/ehp.7499 available via                 salient features of the brevetoxin molecules and
 [Online 10 February 2005]                                                                                 cite the modulating effects of altering the lac-
                                                                                                           tone functionality, of changing the nature of
                                                                                                           the side chain, of inducing additional flexibility
Progress on Toxin Structural                         respectively (Baden 1977; Catterall and Risk          in the middle of the molecule, or combinations
Character                                            1981; Poli et al. 1986). The structure of             thereof [e.g., Jeglitsch et al. (1998); Purkerson
Florida red tide inhalation toxicity has been        PbTx-2 (brevetoxin B) was determined by               et al. (1999)]. All derivatives of natural toxin
documented in the Western literature since           Nakanishi’s group in 1981 to be a trans-fused         examined to date reduce toxicity; that is, no
1844. Earlier accounts from Spanish explor-          polyether ladder toxin with syn relative stereo-      derivative has been produced synthetically that
ers of an “irritating essence” predate 1600.         chemistry across each side of the molecule            is more toxic than PbTx-1 or PbTx-2, the tox-
Past explanations for Florida red tide respira-      (Lin et al. 1981). Additional toxins were struc-      ins reputed to be the parent molecules. In fact,
tory distress have included World War I              turally characterized by comparison with the          it can be argued that all other natural toxins are
nerve gas release (Galtsoff 1948), “fast”- and       PbTx-2 spectroscopic data; PbTx-3 was pro-            produced biosynthetically by K. brevis using the
“slow”-acting toxins (McFarren et al. 1965), a       posed as the reduced form of PbTx-2 by                two parent backbones as precursors.
phosphorylated organic molecule (Martin and          Baden and Mende (1981) and Chou and col-                   Nicoloau et al. (1995) had produced
Chatterjee 1969), and green particles as frag-       leagues (Chou et al. 1985; Shimizu et al.             totally synthetic brevetoxin PbTx-2, which
ments of a marine alga (Woodcock 1948).              1990). Subsequent reduction of PbTx-2 with            had receptor binding properties and spec-
Descriptions of Florida red tide complexity          tritium-labeled sodium borohydride allowed            troscopy identical to the natural material,
have mirrored increases in capability of analyt-     Baden and colleagues to produce isotopically          confirming that the structure proposed by
ical detection. Before 1970, the description of      labeled brevetoxin with sufficient specific           Lin et al. (1981) was indeed correct.
red tides was based on environmental observa-        activity for radioimmunoassays (Baden et al.
tions of irritating aerosols, fish-killing sub-      1984) and later to characterize the brevetoxin
                                                                                                           This article is part of the mini-monograph
stances, and hemolytic factors [reviewed in          binding site in rat brain synaptosome sodium          “Aerosolized Florida Red Tide Toxins (Brevetoxins).”
Baden (1983, 1989)]. Because such small              channels (Poli et al. 1986).                            Address correspondence to D.G. Baden, Center for
amounts of bioactive material were necessary              Shimizu et al. (1986) described                  Marine Science, UNCW, 5600 Marvin Moss Lane,
to elicit the toxicologic responses, much of the     brevetoxin A, possessing a slightly different         Wilmington, NC 28409 USA. Telephone: (910) 962-
initial pharmacology/toxicology was under-           polyether backbone, from cultures of Karenia          2408. Fax: (910) 962-2405. E-mail:
stood long before toxin structures were              brevis. This molecule, denoted PbTx-1, is very          We thank the Harmful Algal Bloom Laboratory for
                                                                                                           Analytical Biotechnology (HABLAB) at UNCW,
known. Investigators in the early 1970s, using       similar to the PbTx-2 structure in that both          technicians S. Niven, J. Lamberto, L. Tomas, and
the new field of high-performance liquid             possess lactone functionality in the A-ring and       E. McConnell, and colleagues C. Tomas and J.L.C.
chromatography, purified two toxins with             a series of relatively rigid rings that form a lad-   Wright.
very similar spectroscopic characteristics, their    der structure, and both terminate in an identi-         Grant support is acknowledged from National
differences being the presence of either an          cal, very reactive α,β-unsaturated aldehyde           Institute of Environmental Health Sciences grants
exomethylene-conjugated aldehyde or the cor-         side chain. It is noteworthy that PbTx-1 is the       R01 ES05853, R01 ES 06411, and P01 ES 10594.
                                                                                                             The authors declare a competing financial interest
responding allylic alcohol. These two materi-        only natural toxin known to possess rings with        concerning brevenal. D.G. Baden and A.J. Bourdelais
als of “interchangeable nature” (T46 and T47,        five, six, seven, eight, and nine members, all in      are named in a patent application for uses of brevenal
or T17 and T34) later became known as the            the same molecule. Seven additional structures        in mucociliary disease treatment.
polyether brevetoxins PbTx-3 and PbTx-2,             followed in quick succession (Figure 1), the            Received 2 August 2004; accepted 9 February 2005.

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Mini-Monograph            |   Baden et al.

Subsequent synthesis of the brevetoxin A                   We restrict ourselves to a discussion of           short-to-long trans-helical peptide regions.
molecule (PbTx-1) was likewise accomplished            those toxins, antagonists, and metabolites that        Three of the helices in each domain are neu-
(Nicoloau et al. 1998). The production of              have been described from seawater or culture           tral in charge; two of the helices are hydro-
truncated brevetoxin by Nicoloau and col-              medium, although we realize that aerosols              phobic, and one helix in each domain is
leagues, a compound possessing all of the              may contain cell particles and organelles              highly positively charged. The highly charged
“required” features of intact brevetoxin except        (Woodcock 1948) as well as bacteria, fungi,            S4 helix in each domain is thought to respond
for the B–E ring spacer region, was essentially        and other organic materials in the mixed               allosterically to changes in membrane poten-
inactive in patch-clamp experiments (Gawley            microlayer of the coastal ocean (Zobell 1942).         tial, a “voltage sensor.” Allosteric realignment
et al. 1995), although it had some affinity for         The metabolites produced by shellfish, fish,           of all four S4 helices results in a change in
receptor site 5 on the sodium channel.                 and other marine creatures, although extremely         channel configuration from “closed” (C), to
     As a result of the multiplicity of effects        important for seafood poisonings, are not con-         “open” or conducting (O). Channels inacti-
demonstrated by this combination of metabol-           sidered to be relevant to inhalation toxicology,       vate or become nonconducting (I) by internal
ically relevant structural modifications, recep-        and they have not been detected by us in air           insertion of the isoleucine–phenylalanine–
tor binding pharmacology, and single-channel           filters (Cheng et al. 2005).                            methionine tripeptide region of the intra-
kinetic studies, it was postulated that Florida            Figure 3 illustrates the model “brevetoxin”        cellular polypeptide connecting domains III
red tide potency in situ would be a complex            that encompasses all the salient features of tox-      and IV into the ion-conducting central pore.
phenomenon and the toxicologic consequences            ins that are “fully active” at their binding site,     Normally, VSSCs open in response to
of red tides would be based on the amounts             voltage-sensitive sodium channels (VSSCs;              membrane depolarization and subsequently
and activities of the brevetoxins present (Baden       membrane potential-gated ion channels)                 inactivate in the late phase after activation.
and Tomas 1988; Steidinger and Baden 1984).            (Baden et al. 1996). Modeling work initiated           Channels return to the allosterically closed
     The purpose of this mini-monograph                by Gawley et al. (1995) described a brevetoxin         configuration during membrane repolarization
introduction is to describe the complex nature         PbTx-2 molecule believed to be composed of             (Taylor 1994). All natural brevetoxins have
of exposure to Florida red tide toxins, as refer-      three distinct segments: a relatively rigid four-      four distinct activities that alter the normal
ence for articles presented later in this mono-        ring H–K ring system, an A-ring lactone, and           C→O→I triad of VSSCs: a) the activation
graph (Figure 2). Although most of the                 a B–G ring region with limited flexibility.            potential is shifted to more negative potentials,
studies have been conducted with PbTx-3 (a             Homologous ring systems exist in the breve-            favoring a C→O allosteric change at normal
natural toxin possessing all of the requisite          toxin A backbone. All active toxins have these
structural characteristics for full activity), the     essential characteristics whether they are mem-          1980—One chemically characterized brevetoxin
investigators acknowledge that many studies            bers of the brevetoxin B backbone or the                      with a specific toxicity
will require repetition for toxins missing one         brevetoxin A backbone (Figure 1).
or more activity loci, or for complex mixtures             Active toxins bind to site 5 on the α-sub-           1985—Multiple characterized brevetoxins,
that may include metabolites and antagonists           unit of the VSSC (Poli et al. 1986) and are                   each with its own specific toxicity
in addition to toxin. Presumably, any bioac-           thought to orient “head-down” into the chan-
tive agents produced by the toxigenic organ-           nel (Gawley et al. 1995), intercalating between
                                                                                                                1990—Multiple brevetoxins, based on
ism or any metabolites free in the water have          the α-helices of domains III and IV of the                    two different structural backbones
the potential to become aerosolized. Further,          VSSC (Trainer et al. 1994) (Figure 4).
we believe that the overall signs and symp-                The α-subunit is a single polypeptide gly-
                                                                                                                1995—Multiple brevetoxins and
toms observed in humans will depend on the             coprotein, which possesses a 4-fold homology.                 active brevetoxin metabolites
particular combination of activities elicited          Each homologous domain contains six trans-
by the toxins and derivatives/metabolites              membrane α-helices (S1–S6), which alter-
                                                                                                                2000—Multiple brevetoxins and metabolites,
(Jeglitsch et al. 1998).                               nately extend through the membrane, with                      modulated by a polyether antagonist brevenal

                                                                                                                2005—Multiple brevetoxins and metabolites,
                                                                                                                     modulated by multiple brevenal structures

                                                                                                              Figure 2. Complexity of lethal components isolated
                                                                                                              from Florida red tide: the explanation for the
                                                                                                              potency of red tide events is summarized from 1980
                                                                                                              to the present. 1981: only one specific toxin had
                                                                                                              been identified. Lethality of red tides was thought
                 Brevetoxin B backbone                                 Brevetoxin A backbone                  to be directly related to the concentration of toxi-
                                                                                                              genic organism and the toxin it produced. Mid-
             Brevetoxin B backbone, R =                              Brevetoxin A backbone, R =               1980s: characterization of multiple toxins with
             PbTx-2, CH2C(=CH2)CHO                                   PbTx-1, CH2C(=CH2)CHO                    widely varying potencies. Lethality was thought to
             PbTx-3, CH2C(=CH2)CH2OH                                 PbTx-7, CH2C(=CH2)CH2OH                  arise from additive effects of each toxin present.
             PbTx-5, [PbTx-2], C-37 OAc                              PbTx-10, CH2CH(CH3)CH2OH                 Early 1990s: the second brevetoxin structural back-
             PbTx-6, [PbTx-2], H-ring epoxide                                                                 bone (PbTx-1) and multiple derivatives discovered
             PbTx-8*, CH2COCH2Cl                                                                              and thought to be more potent than the corre-
             PbTx-9, CH2CH(CH3)CH2OH                                                                          sponding toxins based on the PbTx-2 backbone.
                                                                                                              Early 21st century: brevenal described as the first
Figure 1. Brevetoxins are based on two different structural backbones, based on what are perceived to be      naturally occurring antagonist to counteract the
the two parent molecules, PbTx-2 (brevetoxin B) and PbTx-1 (brevetoxin A). All other known derivatives        effects of brevetoxins in radioligand binding
are based on alteration of the R-side chain, epoxidation across the double bond in the H-ring of PbTx-2, or   assays, in fish and mouse bioassays, and in respi-
derivatization at the C-37 hydroxyl in PbTx-2. PbTx-8, the chloromethyl ketone derivative of PbTx-2, is an    ratory protocols. (The pulmonary receptor for
artifact of chloroform extraction and subsequent phosgene conversion of PbTx-2. Common features               brevetoxins and brevenal may be distinct from the
include trans-fused polyether ring systems consisting of five- to nine-membered rings.                         well-characterized brevetoxin binding site on neu-
*Denotes likely chemical artifact from extraction.                                                            ronal VSSC described in the text.)

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                                                                                    Mini-Monograph             |   Historical complexity of Florida red tide toxins

resting potential; b) a longer mean open                 artifact of the aqueous chloroform extraction                   Cytotoxicity was reported for hemibrevetoxin,
time O, which can be influenced by a number               of PbTx-2, PbTx-13, and PbTx-14 are likely                      but no antagonistic activity was recorded.
factors, but which results in the channel being          artifacts of acid-catalyzed methanol addition                       From the data collected, it is important to
in the open configuration longer; c) an induc-            during purification protocols. All three breve-                  note again that not only are there two struc-
tion of sodium ion subconductance states in              toxin artifacts illustrate the highly reactive                  tural backbones for the brevetoxin molecules,
addition to the normal 21 pS rate; and d) an             nature of the conjugated side chain in the                      but there are at least 13 different derivatives
inhibition of inactivation O→I. Derivatives of           PbTx-2 molecule. By analogy, the side chain                     thereof, and each of these derivatives possesses
natural toxin produced in the laboratory, as             of PbTx-1 ought to be equally reactive. This                    a specific toxicity that is correlated to its bind-
well as metabolites, knock out one or more of            side chain also interacts with nucleophiles                     ing affinity on VSSCs. However, from past
the observed activities in single ion channel            to form adducts: the presumed cathepsin                         work it is clear that derivatives produced at
patch-clamp experiments. The resulting over-             inhibitory activity is thought to reside in the                 specific loci on the brevetoxin backbone impart
all consequences of exposure to multiple nat-            active cysteine of cathepsin B, H, and L and of                 a differential set of “activities” that encompass
ural toxins and metabolites are complex and              papain (Sudaranam et al. 1992). Such activity                   effects on activation potential, mean open
difficult to quantify mechanistically. How               might impart an immune suppression in                           time, inactivation, and persistent depolariza-
metabolites or modifications to toxin loci               exposed individuals (Benson et al. 2005).                       tion due to subconductance state prevalence.
modulate activity is summarized in Table 1.                   PbTx-11, PbTx-12, and PbTx-tbm
     Experimental evidence suggests the H–K              (brevetoxin PbTx-2 lacking the side chain tail)                 Relationship of Toxin Multiplicity
rings system is essential for binding, and all           are new toxins found in cultures and in the                     to Environmental Episodes
toxins and derivatives that are bioactive possess        field (Abraham et al. 2005; Bourdelais and                      of Pulmonary Exposure
this ring system. The B–G ring system is a               Baden 2004; Bourdelais et al., unpublished                      Many of the examined derivatives can be
spacer region that separates binding from an             results). Their toxicology is almost completely                 produced by intermediary metabolism and
activity locus, the A-ring. The intact A-ring            unknown except that they competitively bind                     environmental decomposition, suggesting that
lactone modulates both inactivation activity             at site 5 on the sodium channel (Bourdelais                     environmental episodes may exhibit great vari-
and mean open time in single-channel patch-              and Baden 2004; Bourdelais et al. 2004).                        ation in both degree and effect (Baden and
clamp experiments (Jeglitsch et al. 1998;                Figure 6 shows the chemical structure of                        Tomas 1988; Ingle 1954). The added pres-
Purkerson-Parker et al. 2000). Details of the            brevenal, the antagonistic material isolated                    ence of the brevenals would tend to moderate
molecular pharmacology of brevetoxin and                 from both laboratory cultures and bloom                         the overall consequences. Its waxing and wan-
derivatives are reviewed in Baden and Adams              waters (Bourdelais et al. 2004, in press).                      ing presence during a red tide may provide a
(2000) and summarized in Table 1. We                     Brevenal derivatives have also been detected in                 partial explanation for anecdotal accounts of
believe that, depending on the type of toxin             our cultures. Brevenal resembles hemibreve-                     high toxigenic organism cell counts in the
decomposition that occurs during the declin-             toxin, isolated by Shimizu et al. (1990).                       absence of pronounced fish kills or respiratory
ing phases of a Florida red tide, different
inhalation effects may be evident based on
pharmacophore absence or presence. Detailed
work with bioactive polyethers possessing only
some of the activity characteristics of fully
active toxin have not been carried out, but dif-
ferential inhalation symptoms have been expe-
rienced during active red tides [Pierce RH,
personal communication (15 April 2004);
Backer et al. 2005; Fleming et al. 2005].
     Figure 5 illustrates the recently elucidated
structures of PbTx-11 through PbTx-14
(Bourdelais and Baden 2004; Bourdelais et al.,           Figure 4. (A) A model depicting the sodium-channel α-subunit three-dimensional structure and hypothe-
unpublished results). Like PbTx-8, which is an           sized orientation of brevetoxin “head”-down” between domains III and IV. The view from above (at far left)
                                                         illustrates a possible orientation relative to the α-helices, which are shown as colored circles. (B) Diagram
                                             “Tail”      illustrating the putative triad of VSSC configurations and alterations produced by brevetoxins. All natural
                                                         brevetoxins a) shift the activation potential, favoring the open (O) configuration at normal resting potential
                                                         (thick arrow); b) produce a longer mean open time (O); c) induce subconductance states (O1, O2, etc.); and
                                                         d) inhibit inactivation (O→I). Figure adapted from Gawley et al. (1995), Taylor (1994), Rein et al. (1994a,
                                                         1994b), and Jeglitsch et al. (1998).

                                          region”        Table 1. Effect of toxin modification on the five expressed activities of natural brevetoxins.a
 “Head”                                                  Toxin                         Shift           Subconductance           Longer mean              Inhibit           Toxin
            “Spacer region”                              modification                 activation            statesb               open time            inactivation       antagonist
Figure 3. All active brevetoxins have several requi-     Natural toxins                  Yes                Yes (1)                  Yes                  Yes                No
site features that result in complete expression of      Modified A-ring                  Yes                 No                      No                   Yes                No
brevetoxin activities: a relatively rigid H–K ring       Open A-ring                     Yes                Yes (4)                  Yes                  Yes                No
region (G–J region in PbTx-1) thought to be involved     Short spacer region             Yes                 No                      No                   No                 No
in binding at site 5 of the VSSCs on the α-subunit       Bulky side chain                Yes                 No                      No                   No                 Yes
(“rigid region”); an A-ring electrophile of some type,   Saturated H-ring                Noc                 No                      No                   No                 No
in most cases a five- or six-membered ring lactone        Brevenal                        ND                  ND                      ND                   ND                 Yes
(“head”); a several-ring “spacer” region that sepa-      ND, not determined.
rates the binding region from the activity region;       aCompiled from Baden et al. (1996), Bourdelais et al. (2005, in press), and Gawley et al. (1992). bIn addition to the normal
and a side chain (“tail”; Gawley et al. 1995).           21 pS conductance. cHas no affinity for site 5.

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irritation, or of relatively low cell counts and         date. Moreover, the relative concentrations of           complex combination of acute and chronic
reports of “respiratory irritation” (Fleming             toxin and antagonist in the pulmonary system             signs and symptoms in both animals and
et al. 2005).                                            are reversed from those exhibited by neuronal            humans exposed to aerosolized bioactive mate-
     What is there to say, then, about the over-         systems. That is to say, brevenal is a more              rials produced by K. brevis.
all pulmonary effects of these polyether natural         potent antagonist in pulmonary systems than
products? First, in 1947 Woodcock collected              it is in neuronal or fish bioassays. This is an                                   REFERENCES
droplets of vapor from active red tides and              interesting set of data because it tends to sup-
from normal seawater. The droplets from the              port the contention that there is something              Abraham WM, Bourdelais AJ, Ahmed A, Sereberiakov I, Baden
                                                                                                                      DG. 2005. Effects of inhaled brevetoxins in allergic air-
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from non-red tide areas (Woodcock 1948).                 when compared with site 5 of VSSC in the                     intervention. Environ Health Perspect 113:632–637.
Red tide aerosol droplets contained small,               central nervous system. Either way, further              Backer LC, Kirkpatrick B, Fleming LE, Cheng YS, Pierce R, Bean
                                                                                                                      JA, et al. 2005. Occupational exposure to aerosolized breve-
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                                                                                                                      Tide Dinoflagellate Gymnodinium breve [PhD Thesis].
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                                                                                                                  Baden, DG. 1989. Biochemistry of the brevetoxins: potent acti-
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     As described by Abraham et al. (2005), the          tiple biotoxins and antagonists, and these               Bourdelais AJ, Campbell S, Jacocks H, Naar J, Wright JLC,
brevetoxins have effects at 1,000-fold lower             interact with at least three receptors located in            Carsi J, et al. 2004. Brevenal is a natural inhibitor of breve-
concentrations in pulmonary systems than                 neuronal, pulmonary, and enzymatic regula-                   toxin action in sodium channel receptor binding assays.
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they have in any neuronal system studied to              tory systems of living organisms. The result is a        Bourdelais AJ, Campbell SK, Kubanek J, Wright JLC, Baden DG.
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                                                         a 6–7–6–7–7 ring motif, these materials bind with
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                                                         high affinity in brevetoxin receptor assays, and effec-       Perspect 113:638–643.
                                                         tively act as inhibitors of brevetoxin binding and       Chou HN, Shimizu Y, VanDuyne G, Clardy J. 1985. Isolation and
Figure 5. Five new brevetoxins, based on the PbTx-2      activity. Brevenal is the major constituent derived          structure of two new polycyclic ethers from Gymnodinium
type backbone, have been purified and character-          from cultures or the environment, with smaller               breve Davis (= Ptychodiscus brevis ). Tetrahedron Lett
ized: PbTx-11, a toxin with a shortened side chain;      amounts of brevenol (brevenal with the aldehyde              26:2865–2868.
PbTx-12, the only natural ketone brevetoxin known;       reduced to the alcohol). Reduction of brevenal with      Fleming LE, Kirkpatrick B, Backer LC, Bean JA, Wanner A,
PbTx-13 and PbTx-14, which are both believed to be       tritiated borohydride can produce a new radioligand          Dalpra D, et al. 2005. Initial evaluation of the effects of
extraction artifacts formed in the presence of                                                                        aerosolized Florida red tide toxins (brevetoxins) in persons
                                                         for probing polyether binding sites. Preliminary
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methanol reaction with the very active exomethyl-        experiments indicate high-yield radioligand derivati-    Galtsoff PS. 1948. Red tide. Spec. Sci Rep US Fish Wildl Serv
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a brevetoxin PbTx-2 backbone without any side            brevenal binding to determine if competition is truly    Gawley RE, Rein KS, Jeglitsch G, Adams DJ, Theodorakis EA,
chain, a form that is prevalent in senescent cultures.   competitive awaits completion.

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                                                                                                 Mini-Monograph              |   Historical complexity of Florida red tide toxins

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